1000
J. Nat. Prod. 1996, 59, 1000-1001
Tubercidin Analogs from the Ascidian Didemnum voeltzkowi Scott S. Mitchell,† Steven C. Pomerantz,† Gisela P. Concepcio´n,‡ and Chris M. Ireland*.† Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, and Marine Science Institute, University of the Philippines, Diliman, Quezo´ n City, Philippines Received May 22, 1996X
Two new 5′-deoxypyrrolo[2,3-d]pyrimidine (7-deazapurine) nucleosides, 5′-deoxytubercidin and 5′-deoxy-3-bromotubercidin, were identified from the ascidian Didemnum voeltzkowi. Two known anomers of 5′-deoxy-3-iodotubercidin were also purified from the extract. Assignments were made on the basis of 1H and 13C chemical shifts as well as HPLC-MS experiments. Marine organisms have proven to be a rich source for a wide variety of modified nucleosides.1,2 Pyrrolo[2,3d]pyrimidine (7-deazapurine) nucleosides such as the natural product tubercidin have been reported from microorganisms,3 marine algae, and a marine sponge.4 Kazlauskas et al., Davies et al., and Phillis et al. described the isolation and bioactivity of 5′-deoxy3-iodotubercidin (1) from the red algae Hypnea valendiae.4-6 In the same series of publications, the isolation of the nucleoside base 4-amino-3-bromopyrrolo[2,3-d]pyrimidine from the marine sponge Echinodictyum sp. was reported. We report here the isolation of three tubercidin analogs; 1, 5′-deoxy-3-bromotubercidin (2), and 5′-deoxytubercidin (3) from the ascidian Didemnum voeltzkowi (Savigny, 1816, family Didemnidae). To the best of our knowledge, both compounds 2 and 3 are novel compounds.
The frozen ascidian (400 g wet weight) was extracted with MeOH and the resulting crude extract partitioned according to a modified Kupchan fractionation protocol.7 The CHCl3-soluble material and the aqueous MeOH were then subjected to C18 VLC (vacuum liquid chromatography) and HPLC. Compounds 1 and 2 were purified from the CHCl3-soluble Kupchan fraction while 3 was purified from the aqueous MeOH soluble fraction. The nucleosides thymidine and uracil were also identified as components of the aqueous MeOH Kupchan fraction. The presence of pyrrolo[2,3-d]pyrimidine deoxyribose nucleosides was initially recognized by a fragment in the FABMS at m/z 251 in all three tubercidin analogs. The molecular ion of the brominated compound showed the expected M + 2 isotope pattern for a singly brominated molecule, while the iodinated compound contained * Author to whom correspondence should be addressed. † University of Utah. ‡ University of the Philippines. X Abstract published in Advance ACS Abstracts, September 15, 1996.
S0163-3864(96)00457-0 CCC: $12.00
Table 1.
1H
and
13C
Assignments of Compounds 1 and 3 compd 1
position
13Ca,b
2 3 4 6 8 9 1′ 2′ 3′ 4′ 5′
102.9 49.9 157.2 151.9 127.9 149.5 89.3 77.9 76.4 81.3 13.2
b
3 1H
13C
5.99, d (5.0) 4.37, dd (5.0,5.2) 3.83, dd (5.3,5.3) 3.87, dd (6.5,5.3) 1.26, d (6.5)
123.2 101.2 158.9 152.3 104.0 151.2 89.7 75.8 76.5 80.8 19.2
7.75, s 8.09, s
1H
7.23, d (3.7) 6.64, d (3.7) 8.08, s 6.12, d (4.6) 4.42, dd (4.6,5.5) 3.96, dd (5.5,5.5) 4.06, dq (6.4,5.8) 1.39, d (6.4)
a Chemical shift values from spectra collected in CD OD. 3 Coupling constants reported in Hz.
an M - 126 ion suggesting halogen matrix exchange. The ribose sugar moiety was recognizable in 1 and 3 by characteristic anomeric proton doublets at 5.99 and 6.12 ppm, respectively, in the 1H NMR spectra of the compounds (Table 1). The absence of geminal proton coupling expected for 5′ protons of nucleosides in the 1H NMR of 1 and 3 suggested modification at this site. Doublets at 1.26 and 1.39 ppm that integrate to three protons in the 1H NMR of 1 and 3, respectively, were consistent with 5′ deoxy sugars. The previous publication of 1 from the red algae Hypnea valendiae reported the purification of two isomers of the compound. On the basis of JH1-H2 coupling constants Wells proposed that the two compounds were anomers. We also have evidence for the presence of two chromatographically distinct isomers of 1 from Didemnum voeltzkowi. C18 VLC of the CHCl3 Kupchan fraction yielded a mixture of nucleosides that eluted using CH3CN/H2O (30:70). When this material was subjected to C18 HPLC using CH3CN/0.1 M aqueous NH4OAc (20:80) two distinct peaks eluted at 16 min and 21 min. The first peak appeared pure by NMR spectroscopy and contained one molecular ion at m/z 377 in the FABMS. The 1H NMR spectrum of material isolated as the second peak showed slight doubling of all of the signals in the 1H NMR spectrum, while the FABMS showed peaks at m/z 377 and two peaks of equal intensity at m/z 327 and 329. This suggested that the sample contained a second isomer of 3-iodo-5′deoxytubercidin as well as a brominated analog. HPLC-MS was then employed to further investigate the structure of the brominated compound. Using a modified Buck gradient9 two major peaks were clearly resolved by HPLC. The thermospray mass spectrum
© 1996 American Chemical Society and American Society of Pharmacognosy
Notes
of the first HPLC peak contained ions at m/z 331, 329, 251, 215, 213, and 134. The m/z 331 and 329 peaks corresponded to the molecular ion, and the 251 ion represented loss of a bromine atom with subsequent replacement by a proton. The 215 and 213 pair of ions arose from the loss of a deoxyribose sugar, demonstrating that the molecule is brominated somewhere on the nucleoside base. The mass spectrum of the second HPLC peak contained a molecular ion at m/z 377 representing 1 as well as ions corresponding to the same fragmentation pattern as the brominated compound. The 1H NMR spectrum of the mixture contained singlets of different intensities at 7.78 and 7.71 ppm, assignable to C-2 of the pyrrolopyrimidine base. The observation that these signals were singlets was consistent with halogenation on C-3. Further attempts to separate the compounds involved C18 HPLC with a solvent system of CH3CN/0.05% aqueous trifluoroacetic acid (20:80) resulted in degradation of the compounds. We were unable to further characterize the brominated analog at this point as no more sample was available. 5′-Deoxytubercidin was initially detected in the aqueous MeOH-soluble Kupchan fraction after it eluted from a C18 VLC column using MeOH/H2O (40/60). This material was further purified by using C18 HPLC with a solvent system of CH3CN/0.1 M aqueous NH4OAc adjusted to pH 5 to yield pure compound 3 (10.2 mg). FABMS gave a molecular ion of m/z 251. 1H coupling constants of 3.7 Hz for protons assigned to position 2 and 3 are expected values for the 5-membered aromatic ring. Chemical shifts for 1H and 13C NMR data agree with expected values based on the halogenated analogs. Experimental Section General Experimental Procedures. 1H and 13C NMR experiments were performed on a Varian Unity 500 MHz spectrometer. Spectra were referenced to residual undeuterated solvent peaks or solvent 13C solvent signals. High- and low-resolution FABMS mass measurements were performed on a Finnegan MAT 95 high-resolution gas chromatograph/mass spectrometer. The HPLC thermospray-MS was perfomed on an instrument of in-house design, the details of which have been previously published.8 The HPLC gradient used in the experiment is a slight modification of the gradient developed by Buck, using procedures that have been previously published.9,10 Extraction and Characterization of Nucleosides. The ascidian was collected from isolated rocks scattered throughout shallow tide pools on Apo Reef, Philippines.
Journal of Natural Products, 1996, Vol. 59, No. 10 1001
A voucher specimen has been submitted to the University of the Philippines labeled as GC95-94-10. The frozen sample was ground using a blender and repeatedly extracted with MeOH. The dried extract was resuspended in 200 mL of 90% MeOH-10% H2O. This solution was extracted with hexane, and then an additional 20 mL of H2O was added to the aqueous MeOH fraction. The resulting 30% aqueous MeOH fraction was then extracted with CHCl3. The CHCl3-soluble material was then subjected to a C18 VLC column using 100 cm3 LiChroprep RP18 in a 10 cm diameter column. Fractions were eluted using a step gradient using increasing amounts of CH3CN. All of the halogenated tubercidin analogs eluted in the CH3CN/H2O (70:30) wash. Reversed-phase HPLC (Rainin Dynamax Microsorb 4.6 × 250 mm 5 µm column) of this fraction using CH3CN/0.1 M aqueous NH4OAc adjusted to pH 5 using acetic acid (15:85) yielded pure 1 (2.5 mg) and a mixture of the second anomer of 1 and compound 2 (2.3 mg). A step gradient C18 VLC column using MeOH/H2O solutions was performed on the aqueous MeOH Kupchan fraction. 5′-Deoxytubercidin eluted in the 60% MeOH40% H2O wash. This material was further purified by C18 HPLC using 15% CH3CN-85% 0.1 M NH4OAc adjusted to pH 5 to yield compound 3 (10.2 mg). Acknowledgment. We thank Dr. Elliot Rachlin for FABMS data. This work was supported by NIH Grant No. CA36622. Partial funding for the Varian Unity 500 NMR spectrometer was provided by NIH Grant No. S10 RR06262 (CMI). Continuing support for this facility was provided by NCI Grant No. 5 P30 CA42014. Support for the HPCL-MS facilities has been provided by NIH Grant No. GM 29812. References and Notes (1) Faulkner, D. J. Nat. Prod. Rep. 1995, 12, 223-270 and earlier references cited within. (2) Isono, K. J. Antibiot. 1988, 41, 1711-1739. (3) Suzuki, S.; Matumo, S. J. Antibiot., Ser. A 1957, 10, 201. (4) Kazlauskas, R.; Murphy, P. T.; Wells, R. J.; Baird-Lambert, J. A.; Jamieson, D. D. Aust. J. Chem. 1983, 36, 165-170. (5) Davies, L. P; Baird-Lambert, J.; Marwood, J. F. Biochem. Pharmacol. 1986, 35, 3021-3029. (6) Phillis, J. W.; Smith-Barbour, M. Life Sci. 1993, 53, 497-502. (7) Kupchan, S. M.; Britton, R. W.; Ziegler, M. F.; Siegel, C. W. J. Org. Chem. 1973, 38, 178-179. (8) Edmonds, C. G.; Vestle, N. L.; McCloskey, J. A. Nucleic Acids Res. 1985, 13, 8197-8206. (9) Buck, M.; Connick, M.; Ames, B. N. Anal. Biochem. 1983, 129, 1-13. (10) Pomerantz, S. C; McCloskey, J. A. Methods Enzymol. 1990, 193, 796-824.
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